Cancer remains an important concern for public health, with nearly 1.5 million new cases and 500,000 deaths estimated for 2009. Recently, overactivity of the Hedgehog (HH) signaling pathway has been reported in a large number of human carcinomas, including breast, prostate, and lung cancer. In addition, several studies support the requirement of an active HH pathway for the proliferation, survival and metastasis of malignant cells. HH signaling is therefore an attractive target for therapeutic development and merits further mechanistic investigation in cell culture models to evaluate its role in the development and progression of cancer. In normal HH signaling, exogenous HH ligands bind to cell surface receptors, resulting in a signaling cascade that culminates in the activation of the Gli2 transcription factor. Gli2 promotes transcription of over 130 genes, several with anti-apoptotic and proliferation-promoting activity. Selective regulation of these genes would allow researchers to carefully probe the downstream effects of overactive HH signaling. DNA-binding polyamides are small molecules that represent uniquely effective tools for the investigation of gene-specific transcriptional inhibition. Through a programmable combination of heterocyclic and aliphatic residues, polyamide structures bind the minor groove of DNA with high sequence selectivity and affinity. Upon exogenous addition, polyamides have been shown to regulate gene expression in cancer cell cultures by interfering with transcription factor-DNA interactions in the nucleus. It is thus proposed that the development of a small library of polyamides targeted to genes regulated by Gli2 will allow for the role of HH signaling in cancer to be studied at the gene, genome-wide, and cellular level. The specific aims of this proposal are: 1) to develop a small library of polyamides that interrupt Gli2- DNA interactions in a sequence-specific manner; 2) to investigate the ability of these compounds to alter mRNA expression levels in healthy and tumor cell cultures; and 3) to assess the effect of polyamide transcriptional inhibitors on global genome expression, cell proliferation and cell viability. To begin, we will synthesize six polyamides targeted to cancer-relevant genes and measure their DNA-binding affinity as well as their ability to displace Gli2 from the promoter sequences of these genes in vitro. We will then study the effects of these compounds on the mRNA levels of their target genes, as well as their ability to bind to chromatin- bound DNA, in healthy cells as well as basal cell carcinoma and prostate cancer cell models. The genome wide effects of polyamide transcriptional inhibition will be analyzed using standard microarrays, and the gross cell effects will be measured through microscopy, commercial proliferation assays, and cell sorting. These results will be compared to similar studies with a generic HH pathway inhibitor. The development of Gli2- inhibiting polyamides will provide novel tools for the investigation of HH signaling in cancer and lend insight into the suitability of this pathway for gene-targeted cancer therapy.